CN109580299A - Bone marrow cell chromosome flaking method - Google Patents
Bone marrow cell chromosome flaking method Download PDFInfo
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- CN109580299A CN109580299A CN201710895840.2A CN201710895840A CN109580299A CN 109580299 A CN109580299 A CN 109580299A CN 201710895840 A CN201710895840 A CN 201710895840A CN 109580299 A CN109580299 A CN 109580299A
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- 238000012545 processing Methods 0.000 abstract description 5
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- 238000012360 testing method Methods 0.000 description 24
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- 235000011164 potassium chloride Nutrition 0.000 description 10
- 238000005516 engineering process Methods 0.000 description 8
- 238000000386 microscopy Methods 0.000 description 8
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- 108010047620 Phytohemagglutinins Proteins 0.000 description 5
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/305—Fixative compositions
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- Physics & Mathematics (AREA)
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- Immunology (AREA)
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- Engineering & Computer Science (AREA)
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Abstract
The present invention provides a kind of bone marrow cell chromosome flaking method, and the bone marrow cell chromosome flaking method includes at least: inorganic agent being added into bone marrow cell, its mitosis is stopped, being incubated for；Hyposmosis processing: hypotonic solution being added into bone marrow cell, stands 20~28min；Cell is fixed: fixative being added into bone marrow cell, stands；Film-making, roasting piece；Dyeing.Simpler using preparation method of the present invention, the film-making of acquisition is more clear.
Description
Technical field
The present invention relates to a kind of bone marrow cell chromosome flaking methods, belong to field of biomedicine.
Background technique
Chromosome is the carrier of gene, and karyotype is a group chromosome or a set of chromosome specific to a certain species
Morphological feature, by being chromosomal disorder, hereditary disease, reproducibility to chromosome number purpose technology and to the observation of structure, analysis
The diagnosis of the diseases such as the neoplastic hematologic disorder of genetic alteration provides morphologic basis, for tumor patient curative effect evaluation, Index for diagnosis
With important clinical meaning.
The method of the observation of chromosome is mainly established by nineteen sixty-eight Sweden Casperson, by metaphase chromosome through pre-
Processing, then dyed with different methods, make the articulate fascia for occurring obvious and stable on chromosome.It is broadly divided into two major classes: Yi Leishi
Colored zone is distributed in whole chromosome, such as G, Q, R band；Another kind of is local aobvious band, and a small number of specific regions is made to show bands, as C,
T and N band.This detection scheme is mainly analyzed using G band, is most common banding technique, feature is highly stable.
Marrow is the important hematopoietic tissue of human body.The marrow of adult is divided to two kinds: red marrow and yellow marrow.Red marrow energy
Manufacture red blood cell, blood platelet and various leucocytes.Mitotic index is higher in bone marrow cell, therefore can directly obtain mid-term
Cell therefore only can be so that many be blocked for splitting status cell having silk to the colchicin of doses
Metaphase.
Chromosome specimen preparation is during chromosome karyotype analysis in most important technology and clinical genetics diagnosis
One of common detection method.Stablize and obtain enough division phases, is aobvious with clear, the preferable chromosome of form, there is following key
Factor must be taken into account: (1) distribution situation of chromosome: observing under the microscope, if there is intersection, overlapping etc. between chromosome
Not scattered situation will affect the judgement and analysis of caryogram；(2) band of chromosome: if chromosome bands are bad, such as item
With smudgy, band has hyperchromasia or excessively shallow, influences the interpretation of abnormal results, or even can not judge.
It is thin that traditional bone marrow cell chromosome Slide processing mainly comprises the steps that materials, colchicin block
Born of the same parents' division, Hypotonic treatment make Chromosome spread, pre-fix, cell is fixed, film-making and microscopy.Pre-fix main function and be for
During the low cell harvest of termination, fixed number and time will affect the harvest effect of chromosome.Pre-fix the dosage of liquid
It is final to influence Chromosome spread quality, it is increased with liquid concentration is pre-fixed, the cell the easy to be agglomerating, more difficult point during film-making
It opens, chromosome is easily built up, and production effect is poor.If the hypotonic time is longer, cell rupture is easily caused, chromosome is from cell compartment
It leaks out, causes to lose.
But above method is complex, especially for the control for the dosage and time for pre-fixing liquid, if processing is not
When the quality that will seriously affect film-making.
Summary of the invention
In view of the foregoing deficiencies of prior art, the purpose of the present invention is to provide a kind of bone marrow cell chromosome film-makings
Method is more difficult to control for solving flaking method in the prior art, the undesirable problem of film-making result.
In order to achieve the above objects and other related objects, the present invention provides a kind of bone marrow cell chromosome flaking method, institute
State method the following steps are included:
(1) inorganic agent is added into bone marrow cell stops its mitosis, is incubated for.
Bone marrow cell can come from the myeloid lineage at any position of human body.
Further, the bone marrow cell is the cell after culture.
Preferably, the bone marrow cell is from patient diagnosed, non-patient diagnosed or the trouble under a cloud with certain disease
Person.
Preferably, it when bone marrow cell is from non-patient diagnosed or when the patient under a cloud with certain disease, uses
Culture solution A, 20~48h of incubation time.Simultaneously in order to avoid false positive or false negative can prepare while prepare control group, use
Add the culture solution A (i.e. C culture solution) of stimulant.
Preferably, when the bone marrow cell comes from B cell lymphoma, culture solution A, 70~74h of incubation time are used.Into one
Step ground, in order to avoid false positive or false negative can prepare while prepare control group, using the culture solution A of stuffing polysaccharide (LPS).
Preferably, when coming from t cell lymphoma, the culture solution used selects A culture solution, 70~74h of incubation time.Into one
Step ground, in order to avoid false positive or false negative can prepare while prepare control group, using joined phytohemagglutinin
(PHA) A culture solution.
Further, above-mentioned A culture solution may is that 1640 culture medium: calf serum: mycillin=500:150:7 (body
Product ratio).
Further, above-mentioned C culture solution can be 1640 culture medium: calf serum: mycillin: recombinant human granulocyte
Stimulating factor=500:150:7:0.15 (volume ratio).
Further, any one or a few in colchicinamide, colchicin of the inorganic agent.Preferred
Colchicinamide is used in scheme, the two effect is identical, but colchicinamide toxicity substantially reduces.
Further, the amount that the inorganic agent is added is 80~100 microlitres.
Further, the condition of the incubation is: 36~38 DEG C, 20~40min；Preferably 37 DEG C.
This step processing main purpose is that cell division is made mutually to be maintained at mid-term, convenient for the morphologic observation in later period.
(2) hyposmosis is handled: hypotonic solution being added into bone marrow cell, stands 20~28min.
Further, the solute of the hypotonic solution be selected from KCl (potassium chloride) solution, the concentration of the solution be 0.07~
0.08mol/L, more preferably 0.075mol/L.The KCL solution refers to KCl aqueous solution.
Specific processing method includes: that the bone marrow cell after being incubated in step (1) is centrifuged 1200~1500r/min, from
5~10min of the heart.Supernatant is abandoned, test tube bottom is stirred and mixes the cell of deposited bottom, and the concussion that is vortexed, be added 8~12ml's
KCl hypotonic medium mixes, and stands 20~28min.More preferably 20min..Main purpose is to make Chromosome spread, drawout,
Convenient for later observations.
Further, bone marrow cell and inorganic agent are mixed after hypotonic solution being preheated to 37 DEG C.
It is highly preferred that hypotonic solution first half is slowly added to, latter half gradually accelerates speed addition.It is described slowly to add
Enter to refer to that the mode being drop by drop added is added.
(3) cell is fixed: fixative is added to the bone marrow cell that Hypotonic treatment is crossed is directly middle.
It needs to pre-fix in the bone marrow cell after Hypotonic treatment in the prior art, and the technology in the application
Without being pre-fixed in scheme.
Further, the fixative selects the mixed solution of methanol and glacial acetic acid, further, the methanol and ice vinegar
Acid solution volume ratio 3:1.
Further, the fixative is added in three times, stands after fixative is added every time.
Specific method includes: that the bone marrow cell after standing, 1200~1500r/min are centrifuged 5~10min, abandons supernatant
Liquid stirs test tube bottom and mixes the cell of deposited bottom, and the concussion that is vortexed, the fixative of addition, mixing, it is stored at room temperature 15~
After 20min, 1200~1500r/min is centrifuged 5~10min, abandons supernatant, stirs test tube bottom and mix the cell of deposited bottom
It is even, and the concussion that is vortexed, the fixative of addition mix, after being stored at room temperature 10~15min；1200~1500r/min, centrifugation 5~
10min abandons supernatant, stirs test tube bottom and mixes the cell of deposited bottom, and the concussion that is vortexed, and the fixative of addition mixes,
After being stored at room temperature 10~15min, 1200~1500r/min is centrifuged 5~8min.After cell harvests, it is put into 4 DEG C of -8 DEG C of refrigerators
It saves.
Preferably, the amount that fixative is added for the first time is 8~12ml, it is therefore preferable to 10ml.It is highly preferred that before fixative
Half part is drop by drop slowly added to, and latter half gradually accelerates speed addition.
Preferably, the amount of the fixative of the 2nd time and the 3rd time addition is all 3~7ml, more preferably 5ml.
(4) film-making, roasting piece.
The film-making is by the way of scribing.
Further, the specific method of the scribing includes: hand-held slide one end, and slide is slightly to lower right corner inclination 10~25
Degree, using dropper from the upper left corner along slide top edge to the uniform scribing in the upper right corner.
Further, the roasting piece includes that the slide that will be prepared is put into oven, and first low temperature bakes piece, and high temperature bakes piece again.
Further, the low temperature bakes 55~65 DEG C of piece, 14~18h, more preferably 60 DEG C, 16h.
Further, high temperature bakes 85~95 DEG C of piece, 30~50min, more preferably 90 DEG C, 45min.
(5) it dyes.
Principle: by the processing of proteolytic enzyme, chromosome can be coloured by coloring agents such as Jim Sas and show band, generation depth,
Shallow alternate band.The different band of chromosome is related with corresponding base-pair ingredient.Bathozone is corresponding to be rich in A-T base-pair, has
The gene of function is less.Shallow band is corresponding to be rich in G-C base-pair, and functional gene is more.Every chromosome has its unique G-C band
Mode includes displacement and recombination chromosome so whole chromosome can be helped distinguish between.
Specifically, the method for the dyeing includes: that bone marrow cell is added to coloring agent after proteolysis enzymatic treatment.
Preferably, the proteolytic enzyme is pancreatin；The coloring agent is Giemsa stain.
(6) microscopy.
In the preferred scheme, as far as possible less oil dripping on slide.
As described above, bone marrow cell chromosome flaking method of the invention, has the advantages that
It is fixed in advance in the present invention due to being not necessarily to, and uses special flaking method, so that chromosome in film-making
Morphosis is more clear, and is conducive to microscopy observation.Middle flaking method is more simple compared with the existing technology and film-making is more clear
It is clear.
Detailed description of the invention
Fig. 1 is shown as in embodiment 1 microscopic examination result figure under 1500 times of mirrors of control group.
Fig. 2 is shown as in embodiment 1 microscopic examination result figure under 1500 times of mirrors of experimental group.
Fig. 3 is shown as in embodiment 2 microscopic examination result figure under 1500 times of mirrors of control group.
Fig. 4 is shown as in embodiment 2 microscopic examination result figure under 1500 times of mirrors of experimental group.
Fig. 5 is shown as in embodiment 3 microscopic examination result figure under 1500 times of mirrors of control group.
Fig. 6 is shown as in embodiment 3 microscopic examination result figure under 1500 times of mirrors of experimental group.
Fig. 7 is shown as in embodiment 4 microscopic examination result figure under 1500 times of mirrors of control group.
Fig. 8 is shown as in embodiment 4 microscopic examination result figure under 1500 times of mirrors of experimental group.
Fig. 9 is shown as in embodiment 5 microscopic examination result figure under 1500 times of mirrors of control group.
Figure 10 is shown as in embodiment 5 microscopic examination result figure under 1500 times of mirrors of experimental group.
Specific embodiment
Illustrate embodiments of the present invention below by way of specific specific example, those skilled in the art can be by this specification
Other advantages and efficacy of the present invention can be easily understood for disclosed content.The present invention can also pass through in addition different specific realities
The mode of applying is embodied or practiced, the various details in this specification can also based on different viewpoints and application, without departing from
Various modifications or alterations are carried out under spirit of the invention.The process equipment or device not indicated specifically in embodiment are all made of ability
Conventional equipment or device in domain.Before further describing the specific embodiments of the present invention, it should be appreciated that protection model of the invention
It encloses and is not limited to following specific specific embodiments；It is also understood that term used in the embodiment of the present invention is to retouch
Specific specific embodiment is stated, rather than limiting the scope of protection of the present invention；In description of the invention and claim
In book, unless in addition explicitly pointed out in text, singular "one", " one " and " this " include plural form.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range
Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, the present invention used in all technologies and
Scientific term is identical as the normally understood meaning of those skilled in the art of the present technique.Except specific method, equipment used in embodiment,
Outside material, grasp and record of the invention according to those skilled in the art to the prior art can also be used and this
Any method, equipment and the material of the similar or equivalent prior art of method described in inventive embodiments, equipment, material come real
The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck
Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of related fields.These technologies have perfect explanation in the prior art, and for details, reference can be made to Sambrook etc.
MOLECULAR CLONING:A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN
ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
Material and reagent:
75% alcohol, medical cotton stick, medical cotton ball, LPS, PHA, 25cm2Culture bottle, 15ml centrifuge tube, 10ml pipette,
1ml pipette, slide, 5ml syringe, liquid-transfering gun, micro sample adding appliance, pipette tips, vortex oscillator, centrifuge, Pasteur dropper.
Culture solution: culture solution A, culture solution C, culture solution A+LPS, culture solution A+PHA
Reagent: colchicinamide, colchicin, 0.075mol/L KCl solution, methanol, acetic acid
False negative control experiment is provided in each embodiment below.
Embodiment 1
(1) sample (from Shanghai City hospital) is taken, 2000r/min is centrifuged 10min.Tentatively it is judged as marrow series leukemia.
0.08ml sample leukocytic cream is taken to be seeded in 37 DEG C of A culture solution；37 DEG C of cultures are for 24 hours.Take two 15ml centrifugations
Pipe is added 80 microlitres of colchicinamide, is separately added into bone marrow cell, while sticking sample label, is experimental group and control respectively
Group is incubated for 37 DEG C, 40min.
(2) hyposmosis is handled: hypotonic solution being added into bone marrow cell.
Centrifuge tube 1200r/min is taken out, 10min is centrifuged.Supernatant is abandoned, test tube bottom is stirred and mixes the cell of deposited bottom
It is even, and the concussion that is vortexed, the KCl hypotonic medium (0.075mol/L) of 10ml is added, hypotonic solution first half drop by drop slowly adds
Enter, latter half gradually accelerates speed addition, mixes, and stands 28min.In control group, after standing 40min after addition hypotonic medium
1ml fixer is added to be pre-fixed.
(3) cell is fixed: fixative being added into bone marrow cell.
By centrifuge tube 1500r/min, centrifugation 10min.
Supernatant is abandoned, test tube bottom is stirred and mixes the cell of deposited bottom, and the concussion that is vortexed, be slowly added to consolidating for 10ml
Determine liquid (methanol and glacial acetic acid solution volume ratio 3:1), the first half of fixative is drop by drop slowly added to, and latter half is gradually
Accelerate speed to be added, mix.
After being stored at room temperature 20min, 1200r/min is centrifuged 8min.Supernatant is abandoned, stirs test tube bottom for deposited bottom
Cell mixes, and the concussion that is vortexed, the fixative 5mL of addition, mixes, and stands 15min.1200r/min is centrifuged 8min.Abandon supernatant
Liquid stirs test tube bottom and mixes the cell of deposited bottom, and the concussion that is vortexed, the fixative 5mL of addition are mixed, and are stood
15min.1200r/min is centrifuged 8min.After cell harvests, it is put into 4 DEG C of refrigerators and saves.
(4) film-making, roasting piece.
The film-making uses dicing methods, and control group is by the way of dripping piece.
The slide prepared is put into oven, temperature 60 C, 12h, then 90 DEG C of cellular aging process temperature, 45min.
(5) it dyes.
Baked slide is taken out, is down to room temperature to temperature, adds dye vat (to contain after pancreatin is handled bone marrow cell
Giemsa stain) in.That is pancreatin storing liquid 8 seconds, normal saline flushing twice after, dye 11 minutes, then pure water rinsing.
(6) microscopy, as far as possible less oil dripping on slide.
As a result experimental group such as Fig. 2, control group such as Fig. 1.It can be seen that Fig. 2 form is good, band is clear, and Fig. 1 form is poor, band mould
Paste, Fig. 1 are easier to omit abnormal.
Embodiment 2
(1) sample (from Shanghai City hospital) is taken, 2000r/min is centrifuged 10min.Tentatively it is judged as marrow series leukemia.
0.08ml sample leukocytic cream is taken to be seeded in 37 DEG C of A culture solution；37 DEG C of cultures are for 24 hours.Take two 15ml centrifugations
Pipe is added 90 microlitres of colchicinamide, is separately added into bone marrow cell, while sticking sample label, is experimental group and control respectively
Group is incubated for 37 DEG C, 20min.
(2) hyposmosis is handled: hypotonic solution being added into bone marrow cell.
Centrifuge tube 1300r/min is taken out, 10min is centrifuged.Supernatant is abandoned, test tube bottom is stirred and mixes the cell of deposited bottom
It is even, and the concussion that is vortexed, the KCl hypotonic medium of 10ml is added, (0.07mol/L) hypotonic solution first half drop by drop slowly adds
Enter, latter half gradually accelerates speed addition, mixes, and 20min is stood, in control group, after standing 40min after addition hypotonic medium
1ml fixer is added to be pre-fixed.
(3) cell is fixed: fixative being added into bone marrow cell.
By centrifuge tube 1500r/min, centrifugation 10min.
Experimental group and control group: abandoning supernatant, stirs test tube bottom and mixes the cell of deposited bottom, and the shake that is vortexed
It swings, is slowly added to the fixer (methanol and glacial acetic acid solution volume ratio 3:1) of 10ml, the first half of fixative drop by drop delays
Slow to be added, latter half gradually accelerates speed addition, mixes.
After being stored at room temperature 15min, 1200r/min is centrifuged 8min.Supernatant is abandoned, stirs test tube bottom for deposited bottom
Cell mixes, and the concussion that is vortexed, the fixative 5ml of addition, mixes, and stands 10min.1200r/min is centrifuged 8min.Abandon supernatant
Liquid stirs test tube bottom and mixes the cell of deposited bottom, and the concussion that is vortexed, the fixative of addition mix, and fixed liquid measure is added
For 5mL, 10min, 1200r/min are stood, is centrifuged 8min.After cell harvests, it is put into 4 DEG C of refrigerators and saves.
(4) film-making, roasting piece.
The film-making uses dicing methods, and control group is by the way of dripping piece.
The slide prepared is put into oven, preferably temperature 60 C, 12h, then cellular aging process, preferable temperature
90℃、45min。
(5) it dyes.
Baked slide is taken out, is down to room temperature to temperature, adds dye vat (to contain after pancreatin is handled bone marrow cell
Giemsa stain) in.That is pancreatin storing liquid 8 seconds, normal saline flushing twice after, dye 11 minutes, then pure water rinsing.
(6) microscopy, as far as possible less oil dripping on slide.
As a result experimental group such as Fig. 4, control group such as Fig. 3.It can be seen that Fig. 4 form is good, band is clear, and chromosome is longer, Fig. 3 form
Poor, band is fuzzy, and chromosome is shorter, and Fig. 3 is easier to omit abnormal.
Embodiment 3
(1) sample (from Shanghai City hospital) is taken, 2000r/min is centrifuged 10min.Tentatively it is judged as B cell lymph
Tumor uses A culture solution+LPS.
0.08ml sample leukocytic cream is taken to be seeded in 37 DEG C of C culture solution；37 DEG C of culture 72h.Take two 15ml centrifugations
Pipe is added 100 microlitres of colchicinamide, is separately added into bone marrow cell, while sticking sample label, is experimental group respectively and right
According to group, it is incubated for 37 DEG C, 25min.
(2) hyposmosis is handled: hypotonic solution being added into bone marrow cell.
Centrifuge tube 1500r/min is taken out, 10min is centrifuged.Supernatant is abandoned, test tube bottom is stirred and mixes the cell of deposited bottom
It is even, and the concussion that is vortexed, the KCl hypotonic medium of 8ml is added, (0.08mol/L) hypotonic solution first half is drop by drop slowly added to,
Speed addition is gradually accelerated in back plate part, mixes, and stands 20min.In control group, add after 40min is stood after addition hypotonic medium
Enter 1ml fixer to be pre-fixed.
(3) cell is fixed: fixative being added into bone marrow cell.
By centrifuge tube 1500r/min, centrifugation 7min.
Experimental group and control group: abandoning supernatant, stirs test tube bottom and mixes the cell of deposited bottom, and the shake that is vortexed
It swings, is slowly added to the fixer (methanol and glacial acetic acid solution volume ratio 3:1) of 10ml, the first half of fixative drop by drop delays
Slow to be added, latter half gradually accelerates speed addition, mixes.
After being stored at room temperature 20min, 1200r/min is centrifuged 8min.Supernatant is abandoned, stirs test tube bottom for deposited bottom
Cell mixes, and the concussion that is vortexed, the fixative 5ml of addition, mixes, and stands 12min.1200r/min is centrifuged 8min.Abandon supernatant
Liquid stirs test tube bottom and mixes the cell of deposited bottom, and the concussion that is vortexed, the fixative 5mL of addition are mixed, and are stood
12min, 1200r/min are centrifuged 8min.After cell harvests, it is put into 4 DEG C of refrigerators and saves.
(4) film-making, roasting piece.
The film-making uses dicing methods, and control group is by the way of dripping piece.
The slide prepared is put into oven, preferably temperature 60 C, 12h, then cellular aging process, preferable temperature
90℃、45min。
(5) it dyes.
Baked slide is taken out, is down to room temperature to temperature, adds dye vat (to contain after pancreatin is handled bone marrow cell
Giemsa stain) in.That is pancreatin storing liquid 8 seconds, normal saline flushing twice after, dye 11 minutes, then pure water rinsing.
(6) microscopy, as far as possible less oil dripping on slide.
As a result experimental group such as Fig. 6, control group such as Fig. 5.It can be seen that Fig. 6 form is good, band is clear, well dispersed, Fig. 5 form compared with
Difference, chromosome bands are fuzzy, well dispersed.Fig. 5 is easier to omit abnormal
Embodiment 4
(1) sample (from Shanghai City hospital) is taken, 2000r/min is centrifuged 10min.Tentatively it is judged as T cell lymph
Tumor.
In the A culture solution+PHA for taking 0.08ml sample leukocytic cream to be seeded in 37 DEG C；37 DEG C of culture 72h.Take two 15ml
Centrifuge tube be added 85 microlitres of colchicinamide, be separately added into bone marrow cell, while sticking sample label, be respectively experimental group and
Control group is incubated for 37 DEG C, 30min.
(2) hyposmosis is handled: hypotonic solution being added into bone marrow cell.
Centrifuge tube 1400r/min is taken out, 10min is centrifuged.Supernatant is abandoned, test tube bottom is stirred and mixes the cell of deposited bottom
It is even, and the concussion that is vortexed, the KCl hypotonic medium (0.075mol/L) of 12ml is added, hypotonic solution first half drop by drop slowly adds
Enter, speed addition is gradually accelerated in back plate part, mixes, and stands 25min.In control group, after standing 40min after addition hypotonic medium
1ml fixer is added to be pre-fixed.
(3) cell is fixed: fixative being added into bone marrow cell.
By centrifuge tube 1500r/min, centrifugation 5min.
Experimental group and control group: abandoning supernatant, stirs test tube bottom and mixes the cell of deposited bottom, and the shake that is vortexed
It swings, is slowly added to the fixer (methanol and glacial acetic acid solution volume ratio 3:1) of 10ml, the first half of fixative drop by drop delays
Slow to be added, latter half gradually accelerates speed addition, mixes.
After being stored at room temperature 15min, 1200r/min is centrifuged 8min.Supernatant is abandoned, stirs test tube bottom for deposited bottom
Cell mixes, and the concussion that is vortexed, the fixative 5ml of addition, mixes, and stands 13min.1200r/min is centrifuged 8min.Abandon supernatant
Liquid stirs test tube bottom and mixes the cell of deposited bottom, and the concussion that is vortexed, the fixative 5mL of addition stand 13min,
1200r/min is centrifuged 8min.After cell harvests, it is put into 4 DEG C of refrigerators and saves.
(4) film-making, roasting piece.
The film-making uses dicing methods, and control group is by the way of dripping piece.
The slide prepared is put into oven, preferably temperature 60 C, 12h, then cellular aging process, preferable temperature
90℃、45min。
(5) it dyes.
Baked slide is taken out, is down to room temperature to temperature, adds dye vat (to contain after pancreatin is handled bone marrow cell
Giemsa stain) in.That is pancreatin storing liquid 8 seconds, normal saline flushing twice after, dye 11 minutes, then pure water rinsing.
(6) microscopy, as far as possible less oil dripping on slide.
As a result experimental group such as Fig. 8, control group such as Fig. 7.It can be seen that Fig. 8 form is good, band is clear, and Fig. 7 form is poor, band mould
Paste, occasionally there is chromosome bifurcation.Fig. 7 is easier to omit abnormal
Embodiment 5
(1) sample (from Shanghai City hospital) is taken, 2000r/min is centrifuged 10min.Tentatively it is judged as marrow series leukemia.
0.08ml sample leukocytic cream is taken to be seeded in 37 DEG C of A culture solution；37 DEG C of cultures are for 24 hours.Take two 15ml centrifugations
Pipe is added 80 microlitres of colchicinamide, is separately added into bone marrow cell, while sticking sample label, is experimental group and control respectively
Group is incubated for 37 DEG C, 40min.
(2) hyposmosis is handled: hypotonic solution being added into bone marrow cell.
Centrifuge tube 1200r/min is taken out, 10min is centrifuged.Supernatant is abandoned, test tube bottom is stirred and mixes the cell of deposited bottom
It is even, and the concussion that is vortexed, the KCl hypotonic medium (0.075mol/L) of 10ml is added, hypotonic solution first half drop by drop slowly adds
Enter, latter half gradually accelerates speed addition, mixes, and stands 20min.In control group, after standing 40min after addition hypotonic medium
1ml fixer is added to be pre-fixed.
(3) cell is fixed: fixative being added into bone marrow cell.
By centrifuge tube 1500r/min, centrifugation 10min.
Experimental group and control group: abandoning supernatant, stirs test tube bottom and mixes the cell of deposited bottom, and the shake that is vortexed
It swings, is slowly added to the fixer (methanol and glacial acetic acid solution volume ratio 3:1) of 10ml, the first half of fixative drop by drop delays
Slow to be added, latter half gradually accelerates speed addition, mixes.
After being stored at room temperature 20min, 1200r/min is centrifuged 8min.Supernatant is abandoned, stirs test tube bottom for deposited bottom
Cell mixes, and the concussion that is vortexed, the fixative 5ml of addition, mixes, and stands 10min.1200r/min is centrifuged 8min.Abandon supernatant
Liquid stirs test tube bottom and mixes the cell of deposited bottom, and the concussion that is vortexed, the fixative of addition mix, and fixed liquid measure is added
For 5mL, 10min, 1200r/min are stood, is centrifuged 8min.After cell harvests, it is put into 4 DEG C of refrigerators and saves.
(4) film-making, roasting piece.
The film-making uses dicing methods, and control group is by the way of dripping piece.
The slide prepared is put into temperature 60 C in oven, 12h, then cellular aging process, 90 DEG C of preferable temperature,
45min。
(5) it dyes.
Baked slide is taken out, is down to room temperature to temperature, adds dye vat (to contain after pancreatin is handled bone marrow cell
Giemsa stain) in.That is pancreatin storing liquid 8 seconds, normal saline flushing twice after, dye 11 minutes, then pure water rinsing.
(6) microscopy, as far as possible less oil dripping on slide.
As a result experimental group such as Figure 10, control group such as Fig. 9.It can be seen that Figure 10 is well dispersed, depth counterband tape is clear, Fig. 9 dispersion
Good, band is relatively fuzzy.Fig. 9 is easier to omit abnormal.
Above embodiment is can not to be interpreted as in order to illustrate embodiment disclosed by the invention to limit of the invention
System.In addition, in various modifications and invention listed herein method, composition variation, do not departing from the scope of the present invention
Be obvious for those skilled in the art under the premise of spirit.Although having combined of the invention a variety of specific
Preferred embodiment has carried out specific description to the present invention, it is to be understood that, the present invention should not be limited only to these specific embodiments.
In fact, various obviously modify as described above for those skilled in the art to obtain invention all should include
Within the scope of the invention.
Claims (10)
1. a kind of bone marrow cell chromosome flaking method, which is characterized in that the bone marrow cell chromosome flaking method at least wraps
It includes:
(1) inorganic agent is added into bone marrow cell stops its mitosis, is incubated for；
(2) Hypotonic treatment: hypotonic solution being added into bone marrow cell, stands 20~28min；
(3) cell is fixed: being directly added into fixative into the bone marrow cell that Hypotonic treatment is crossed, is stood；
(4) film-making, roasting piece；
(5) it dyes.
2. bone marrow cell chromosome flaking method according to claim 1, it is characterised in that: the inorganic agent is selected from autumn waters -- limid eyes
Any one or a few in celestial amide, colchicin.
3. bone marrow cell chromosome flaking method according to claim 1, it is characterised in that: the amount that the inorganic agent is added
It is 80~100 microlitres.
4. bone marrow cell chromosome flaking method according to claim 1, it is characterised in that: it is described it is hypotonic it is molten for KCl it is molten
Liquid.
5. bone marrow cell chromosome flaking method according to claim 1, it is characterised in that: the hypotonic solution first half
Divide and be slowly added to, latter half gradually accelerates speed addition.
6. bone marrow cell chromosome flaking method according to claim 1, it is characterised in that: the fixative selects methanol
With the mixed solution of glacial acetic acid.
7. bone marrow cell chromosome flaking method according to claim 1, it is characterised in that: the fixative adds in three times
Enter, is stood after fixative is added every time.
8. bone marrow cell chromosome flaking method according to claim 7, it is characterised in that: fixative is added for the first time
Amount is 8~12ml；The amount of the fixative of 2nd time and the 3rd time addition is all 3~7ml.
9. bone marrow cell chromosome flaking method according to claim 1, it is characterised in that: the roasting piece includes that will prepare
Good slide is put into oven, and first low temperature bakes piece, and high temperature bakes piece again.
10. bone marrow cell chromosome flaking method according to claim 1, it is characterised in that: the film-making uses scribing
Mode.
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| CN110658046A (en) * | 2019-10-29 | 2020-01-07 | 广州达安临床检验中心有限公司 | Chromosome C banding method |
| CN116399673A (en) * | 2022-12-30 | 2023-07-07 | 苏州药明康德新药开发有限公司 | Cell processing method for optimizing genetic toxicology chromosome aberration test |
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